Compositions and Methods of Delivering Treatments for Latent Viral Infections

ABSTRACT

The invention provides delivery methods and compositions for antiviral therapeutics. Methods and compositions are provided for targeted delivery of antiviral therapeutics into cells of interest using, for example, viral vectors such as adenovirus, AAV, and replication incompetent HSV. These and other delivery systems can be used as vehicles to deliver DNA vectors encoding a nuclease or a cell-killing gene. These delivery methods can also be used to deliver naked DNA or RNA, protein products, plasmids containing a promoter that is active only in a latent viral state which drives a cell-killing gene, or other therapeutic agents.

CROSS-REFERENCE TO RELATED APPLICATION(S)

The present application is a continuation of U.S. Ser. No 14/725,943filed May 29, 2015 (Allowed); which claims priority to, and the benefitof, both U.S. Provisional Patent Application Ser. Nos. 62/005,395 filedMay 30, 2014, and 62/029,072 filed Jul. 25, 2014, the contents of whichare incorporated by reference in their entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under contractsCA139490, CA151459, HL099995, and HL099999 awarded by the NationalInstitutes of Health. The Government has certain rights in theinvention.

FIELD OF THE INVENTION

The invention relates to delivering therapeutics to virus-infectedcells.

BACKGROUND

Viral infections are a significant medical problem. For example, herpesis a widespread human pathogen, with more than 90% of adults having beeninfected. Due to latency, once infected, a host carries the herpes virusindefinitely, even when not expressing symptoms. Similarly, humanpapillomavirus, or HPV is a common virus in the human population, wheremore than 75% of people will be infected. A particular problem is thatviral infections may lead to cancer. For example, integration of HPVinto host DNA is known to result in cancer, specifically cervicalcancer. The Epstein-Barr virus (EBV) not only causes infectiousmononucleosis (glandular fever), but is also associated with cancerssuch as Hodgkin's lymphoma and Burkitt's lymphoma.

Efforts are made to develop drugs that target viral proteins but thoseefforts have not been wholly successful. For example, where a virus isin a latent state, not actively expressing its proteins, there isnothing to target. Additionally, any effort to eradicate a viralinfection is not useful if it interferes with host cellular function.For example, an enzyme that prevents viral replication is not helpful ifit interferes with genome replication in cells throughout the host.

SUMMARY

The invention provides methods and therapeutics for selectively treatinga viral infection in host cells that are infected by the virus. Thetreatment can include causing the death of host cells but only thosecells that are infected. For example, the treatment can includedelivering a gene for a protein that causes cell death, where the geneis under control of a viral regulatory element such as a promoter fromthe genome of the infecting virus or the gene is encoded in a vectorthat includes a viral origin of replication. Where the virus is present,the gene will be expressed and the gene product will cause the death ofthe cell. The gene can code for a protein important in apoptosis, or thegene can code for a nuclease that digests the host genome.

Alternatively, the treatment can include an antiviral therapeutic thatremoves the viral infection without interfering with host cell function.For example, the treatment may include a targetable nuclease that istargeted to viral nucleic acid. The targetable nuclease can be providedin a gene that is under the control of a viral regulatory element suchas a viral promoter or an origin of replication. The invention providesfor targeted delivery of antiviral therapeutics into cells of interestusing, for example, viral vectors such as adenovirus, AAV, andreplication incompetent HSV. These and other delivery systems can beused as vehicles to deliver nucleic acid (DNA, RNA, synthetic nucleicacids, such as PNA, LNA, etc) vectors encoding a nuclease or a cytotoxicgenetic cassette. Delivery methods of the invention are useful todeliver vectors containing antiviral gene editing sequences. Theinvention also contemplates delivering naked DNA or RNA, proteinproducts, plasmids containing a promoter or other regulatory sequencethat is active only in a latent viral state which controls acell-killing genetic construct, or expression of a therapeutic agent(e.g., a cytotoxic protein).

In certain aspects, the invention provides a composition for treating aviral infection. The composition includes a vector that includes a genefor a therapeutic and a sequence that causes the therapeutic to beexpressed within a cell that is infected by a virus. The sequence may bea regulatory element (e.g., a promoter and an origin of replication)from the genome of the virus.

The therapeutic may provide a mechanism that selectively causes death ofvirus-infected cells. In certain embodiments, the therapeutic comprisesa protein that causes death of virus-infected cells. The therapeutic maybe a protein that selectively causes the death of the virus-infectedcells. For example, a protein may be used that restores a deficientapoptotic pathway in the cell. The gene may be, for example, BAX, BAK,BCL-2, or alpha-hemolysin. Preferably, the therapeutic induces apoptosisin the cell that is infected by the virus and does not induce apoptosisin an uninfected cell.

In some embodiments, the therapeutic is a targetable nuclease and thesequence is from a genome of the virus. For example, the targetablenuclease may be cas9 endonuclease and the vector may further encode aplurality of guide RNAs. The guide RNAs may be designed to target thegenome of the cell that is infected by the virus.

In certain embodiments, the promoter only causes the therapeutic to beexpressed within a cell that is in a state of latent infection by thevirus.

The vector may be a viral vector such as, for example, a retrovirus,lentivirus, adenovirus, herpesvirus, poxvirus, alphavirus, vacciniavirus, or adeno-associated viruses. The vector may include a plasmid.The vector may include a nanoparticle, cationic lipids, a cationicpolymers, a metallic nanoparticle, a nanorod, a liposome, a micelle, amicrobubble, a cell-penetrating peptide, or a liposphere.

In some aspects, the invention provides a composition for treating aviral infection. The composition includes a vector that includes anantiviral therapeutic and a regulatory element that causes thetherapeutic to be active within a cell that is infected by a virus. Theantiviral therapeutic may optionally be a gene for a targetable nuclease(e.g., cas9, ZFN, TALENS, a meganuclease) and the regulatory element maybe from a genome of the virus (e.g., a promoter or an origin ofreplication). The vector may encode a guide RNA that targets thenuclease to nucleic acid from a genome of the virus. In certainembodiments, the guide RNA is designed to have no perfect match in ahuman genome. The guide RNAs may target the nuclease to a regulatoryelement in the genome of the virus. Any suitable virus may be treatedsuch as Adenovirus, Herpes simplex, type 1, Herpes simplex, type 2,Varicella-zoster virus, Epstein-Barr virus, Human cytomegalovirus, Humanherpesvirus, type 8, Human papillomavirus, BK virus, JC virus, Smallpox,Hepatitis B virus, Human bocavirus, Parvovirus B19, Human astrovirus,Norwalk virus, coxsackievirus, hepatitis A virus, poliovirus,rhinovirus, Severe acute respiratory syndrome virus, Hepatitis C virus,yellow fever virus, dengue virus, West Nile virus, Rubella virus,Hepatitis E virus, Human immunodeficiency virus (HIV), Influenza virus,Guanarito virus, Junin virus, Lassa virus, Machupo virus, Sabia virus,Crimean-Congo hemorrhagic fever virus, Ebola virus, Marburg virus,Measles virus, Mumps virus, Parainfluenza virus, Respiratory syncytialvirus, Human metapneumovirus, Hendra virus, Nipah virus, Rabies virus,Hepatitis D, Rotavirus, Orbivirus, Coltivirus, or Banna virus.

Certain embodiments of the invention make use of a CRISPR/Cas9 nucleaseand guide RNA (gRNA) that together target and selectively edit ordestroy viral genomic material. The CRISPR (clustered regularlyinterspaced short palindromic repeats) is a naturally-occurring elementof the bacterial immune system that protects bacteria from phageinfection. The guide RNA localizes the CRISPR/Cas9 complex to a viraltarget sequence. Binding of the complex localizes the Cas9 endonucleaseto the viral genomic target sequence causing breaks in the viral genome.In a preferred embodiment, the guide RNA is designed to target multiplesites on the viral genome in order to disrupt viral nucleic acid andreduce the chance that it will functionally recombine.

The invention provides methods for targeted delivery of CRISPR/gRNA/Cas9complex or other therapeutic agents into a cell (including entiretissues) that is infected by a virus. The CRISPR/gRNA/Cas9 complexes ofthe invention can be delivered by viral, non-viral or other vectors.Viral vectors include retrovirus, lentivirus, adenovirus, herpesvirus,poxvirus, alphavirus, vaccinia virus or adeno-associated viruses.Delivery can also be accomplished by non-viral vectors, such asnanoparticles, cationic lipids, cationic polymers, metallicnanoparticles, nanorods, liposomes, micelles, microbubbles,cell-penetrating peptides, or lipospheres. Some non-viral vectors may becoated with polyethyleneglycol (PEG) to reduce the opsonization andaggregation of non-viral vectors and minimize the clearance by thereticuloendothelial system, leading to a prolonged circulation lifetimeafter intravenous administration. Aspects of the invention provide forthe application of energy to delivery vectors for increasedtissue-permeabilizing effects (e.g., ultrasound). The inventioncontemplates both systemic and localized delivery.

Aspects of the invention allow for CRISPR/gRNA/Cas9 complexes to bedesigned to target viral genomic material and not genomic material ofthe host. Latent viruses may be, for example, human immunodeficiencyvirus, human T-cell leukemia virus, Epstein-Barr virus, humancytomegalovirus, human herpesviruses 6 and 7, herpes simplex virus types1 and 2, varicella-zoster virus, measles virus, or human papovaviruses.Aspects of the invention allow for CRISPR/gRNA/Cas9 complexes to bedesigned to target any virus, latent or active.

The presented methods allow for viral genome editing or destruction,which results in the inability of the virus to proliferate and/orinduces apoptosis in infected cells, with no observed cytotoxicity tonon-infected cells. Aspects of the invention involve providing aCRISPR/gRNA/Cas9 complex that selectively targets viral genomic material(DNA or RNA), delivering the CRISPR/gRNA/Cas9 complex to a cellcontaining the viral genome, and cutting the viral genome in order toincapacitate the virus. The presented methods allows for treatmenttargeted disruption of viral genomic function or, in a preferredembodiment, digestion of viral nucleic acid via multiple breaks causedby targeting multiple sites for endonuclease action in the viral genome.Aspects of the invention provide for transfection of a CRISPR/gRNA/Cas9complex cocktail to completely suppress cell proliferation and/or induceapoptosis in infected cells. Additional aspects and advantages of theinvention will be apparent upon consideration of the following detaileddescription thereof.

Aspects of the invention provide a composition for treating a viralinfection. The composition includes a vector comprising a gene for anuclease, a sequence that targets the nuclease to a genome of a virus,and a promoter that promotes transcription from the vector within cellsof a specific type. The composition may be used to treat an infection bya varicella zoster virus, i.e., may be used to treat or prevent shinglesor postherpetic neuralgia. In some embodiments, the cells are nervecells and the promoter causes the expression of the genes selectivelywithin the nerve cells. The promoter may be, for example, acytomegalovirus promoter, a Rous sarcoma virus promoter, or aplatelet-derived growth factor (PGDF) promoter. In certain embodiments,the virus is a varicella zoster virus. The sequence may be designed totarget a regulatory element in the genome of the virus and preferablylacks any exact match in a human genome. The nuclease may be a cas9endonuclease. In some embodiments, the sequence is within a clusteredregularly interspaced short palindromic repeats (CRISPR) region withinthe vector, and the CRISPR region encodes a plurality of guide RNAs thatmatch a plurality of targets within the genome of the virus. A promotermay be used that promotes transcription within the peripheral nervoussystem. Any suitable vector such as an adenoviral vector, a rAAV-basedvector, or a plasmid may be used.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C represent EBV-targeting CRISPR/Cas9 designs. (FIG. 1A)Scheme of CRISPR/Cas plasmids, adapted from Cong L et al. (2013)Multiplex Genome Engineering Using CRISPR/Cas Systems. Science339:819-823. (FIG. 1B) Effect of oriP on transfection efficiency in Rajicells. Both Cas9 and Cas9-oriP plasmids have a scrambled guide RNA.(FIG. 1C) CRISPR guide RNA targets along the EBV reference genome.Green, red and blue represent three different target sequencecategories.

FIGS. 2A-2F represent CRISPR/Cas9 induced large deletions. (FIG. 2A)Genome context around guide RNA sgEBV2 and PCR primer locations. (FIG.2B) Large deletion induced by sgEBV2. Lane 1-3 are before, 5 days after,and 7 days after sgEBV2 treatment, respectively. (FIG. 2C) Genomecontext around guide RNA sgEBV3/4/5 and PCR primer locations. (FIG. 2D)Large deletions induced by sgEBV3/5 and sgEBV4/5. Lane 1 and 2 are 3F/5RPCR amplicons before and 8 days after sgEBV3/5 treatment. Lane 3 and 4are 4F/5R PCR amplicons before and 8 days after sgEBV4/5 treatment.(FIG. 2E and F) Sanger sequencing confirmed genome cleavage and repairligation 8 days after sgEBV3/5 (FIG. 2E) and sgEBV4/5 (FIG. 2F)treatment. Blue and white background highlights the two ends beforerepair ligation.

FIGS. 3A-3M represent cell proliferation arrest with EBV genomedestruction. (FIG. 3A) Cell proliferation curves after different CRISPRtreatments. Five independent sgEBV1-7 treatments are shown here. (FIGS.3B-D) Flow cytometry scattering signals before (FIG. 3B), 5 days after(FIG. 3C) and 8 days after (FIG. 3D) sgEBV1-7 treatments. (FIG. 3E-G)Annexin V Alexa647 and DAPI staining results before (FIG. 3E), 5 daysafter (FIG. 3F) and 8 days after (FIG. 3G) sgEBV1-7 treatments. Blue andred correspond to subpopulation P3 and P4 in (FIGS. 3B-D). (FIGS. 3H andI) Microscopy revealed apoptotic cell morphology after sgEBV1-7treatment. (FIGS. 3J-M) Nuclear morphology before (FIG. 3J) and after(FIGS. 3K-M) sgEBV1-7 treatment.

FIGS. 4A-4E represent EBV load quantitation after CRISPR treatment.(FIG. 4A) EBV load after different CRISPR treatments by digital PCR.Cas9 and Cas9-oriP had two replicates, and sgEBV1-7 had 5 replicates.(FIGS. 4B and C) Microscopy of captured single cells for whole-genomeamplification. (FIG. 4D) Histogram of EBV quantitative PCR Ct valuesfrom single cells before treatment. (FIG. 4E) Histogram of EBVquantitative PCR Ct values from single live cells 7 days after sgEBV1-7treatment. Red dash lines in (FIG. 4D) and (FIG. 4E) represent Ct valuesof one EBV genome per cell.

FIG. 5 represents SURVEYOR assay of EBV CRISPR. Lane 1: NEB 100bpladder; Lane 2: sgEBV1 control; Lane 3: sgEBV1; Lane 4: sgEBV5 control;Lane 5: sgEBV5; Lane 6: sgEBV7 control; Lane 7: sgEBV7; Lane 8: sgEBV4.

FIG. 6 shows CRISPR cytotoxicity test with EBV-negative Burkitt'slymphoma DG-75.

FIG. 7 represents CRISPR cytotoxicity test with primary human lungfibroblast IMR-90.

FIG. 8 shows the use of ZFNs.

FIG. 9 diagrams a method of the invention.

FIG. 10 is a map of an HBV genome.

FIG. 11 shows the results of delivering a viral treatment.

FIG. 12 shows a composition according to certain embodiments.

DETAILED DESCRIPTION

The invention generally relates to compositions and methods for deliveryof therapies targeting viral infection. FIG. 9 diagrams a method of theinvention. FIG. 12 shows a composition according to certain embodiments.In some embodiments, the composition includes a vector such as a plasmidthat includes at least a gene for a therapeutic (e.g., nuclease orapoptosis-related gene) and a viral-driven promoter as shown in FIG. 12.The composition may optionally include one or more of a guide RNA, otherpromoters, replication origin, others, or a combination thereof. Thisinvention provides methods and compositions that to allow effectivedelivery of nucleases or other cytotoxic elements to cells of interest.Methods and compositions are provided for targeted delivery of antiviraltherapeutics into cells of interest using, for example, viral vectorssuch as adenovirus, AAV, and replication incompetent HSV. These andother delivery systems can be used as vehicles to deliver DNA vectorsencoding a nuclease or a cell-killing gene. These delivery methods canalso be used to deliver naked DNA or RNA, protein products, plasmidscontaining a promoter that is active only in a latent viral state whichdrives a cell-killing gene, or other therapeutic agents. Methods andcompositions of the invention are designed to specifically target virusand virus-infected cells.

One of the treatments contemplated by the invention is the use ofnucleases to target viral genomes. In some embodiments, the inventioninvolves delivering a nuclease into a cell of interest. Nucleases havethe ability to incapacitate or disrupt latent viruses within a cell bysystematically causing deletions in the viral genome, thereby reducingthe ability for the viral genome to replicate itself. In embodiments,the treatment comprises CRISPR/Cas and guided RNA complexes, which causeinsertions, deletions, or rearrangements within the viral genome inorder to incapacitate or destroy the virus.

The invention generally relates to compositions and methods forselectively treating viral infections using a guided nuclease system.Methods of the invention are used to incapacitate or disrupt viralnucleic acid within a cell through nuclease activity such as single- ordouble-stranded breaks, cleavage, digestion, or editing. Methods of theinvention may be used for systematically causing large or repeateddeletions in the genome, reducing the probability of reconstructing thefull genome.

i. Treating Infected Cell

FIG. 9 diagrams a method of treating a cell infected with a virus.Methods of the invention are applicable to in vivo treatment of patientsand may be used to treat any cell infected with a virus such as byinitiating apoptosis in the infected cells or by digesting genes ofvirus associated with a latent viral infection. Methods may be used invitro, e.g., to prepare or treat a cell culture or cell sample. Whenused in vivo, the cell may be any suitable germ line or somatic cell andcompositions of the invention may be delivered to specific parts of apatient's body or be delivered systemically. If delivered systemically,it may be preferable to include within compositions of the inventiontissue-specific promoters. For example, if a patient has a latent viralinfection that is localized to the liver, hepatic tissue-specificpromotors may be included in a plasmid or viral vector that codes for atargeted nuclease.

ii. Therapeutic

Methods and compositions of the invention can be used to selectivelycause the death of cells that are infected or to selectively target avirus without interfering with the infected cell.

I. Apoptotic

In some embodiments, the invention provides methods and therapeuticsthat can be used to cause the death of host cells but only those cellsthat are infected. For example, the treatment can include delivering agene for a protein that causes cell death, where the gene is undercontrol of a viral regulatory element such as a promoter from the genomeof the infecting virus or the gene is encoded in a vector that includesa viral origin of replication. Where the virus is present, the gene willbe expressed and the gene product will cause the death of the cell. Thegene can code for a protein important in apoptosis, or the gene can codefor a nuclease that digests the host genome.

The therapeutic may be provided encoded within a vector, in which thevector also encodes a sequence that causes the therapeutic to beexpressed within a cell that is infected by a virus. The sequence may bea regulatory element (e.g., a promoter and an origin of replication)from the genome of the virus. The therapeutic may provide a mechanismthat selectively causes death of virus-infected cells. For example, aprotein may be used that restores a deficient apoptotic pathway in thecell. The gene may be, for example, BAX, BAK, BCL-2, or alpha-hemolysin.Preferably, the therapeutic induces apoptosis in the cell that isinfected by the virus and does not induce apoptosis in an uninfectedcell.

In cases where a small number of cells are infected and it would sufficeto ablate the entire cell (as well as the latent viral genome), anaspect of the invention contemplates administration of a vectorcontaining a promoter which is active in the latent viral state, whereinthe promoter drives a cell-killing gene. HSV is a particularlyinteresting target for this approach as it has been estimated that onlythousands to tens of thousands neurons are latently infected. SeeHoshino et al., 2008, The number of herpes simplex virus-infectedneurons and the number of viral genome copies per neuron correlate withlatent viral load in ganglia, Virology 372(1):56-63, incorporated byreference. Examples of cell-killing genes include apoptosis effectorssuch as BAX and BAK and proteins that destroy the integrity of the cellor mitochondrial membrane, such as alpha hemolysin. (Bayles, “Bacterialprogrammed cell death: making sense of a paradox,” Nature ReviewsMicrobiology 12 pp.63-69 (2014)). Having a promoter that is onlyactivated in latently infected cells could be used not only in thiscontext but also be used to increase selectivity of nuclease therapy bymaking activity specific to infected cells; an example of such apromoter is Latency-Associated Promoter 1, or “LAP1”. (Preston andEfstathiou, “Molecular Basis of HSV Latency and Reactivation”, in HumanHerpesviruses: Biology, Therapy and Immunoprophylaxis 2007.)

In some embodiments, the invention provides a composition that includesa viral vector, plasmid, or other coding nucleic acid that encodes atleast one gene that promotes apoptosis and at least one promoterassociated a viral genome. Apoptosis regulator Bcl-2 is a family ofproteins that govern mitochondrial outer membrane permeabilization(MOMP) and include pro-apoptotic proteins such as Bax, BAD, Bak, Bok,Bcl-rambo, Bcl-xs and BOK/Mtd.

Apoptosis regulator BAX, also known as bcl-2-like protein 4, is aprotein that in humans is encoded by the BAX gene. BAX is a member ofthe Bcl-2 gene family. This protein forms a heterodimer with BCL2, andfunctions as an apoptotic activator. This protein is reported tointeract with, and increase the opening of, the mitochondrialvoltage-dependent anion channel (VDAC), which leads to the loss inmembrane potential and the release of cytochrome c.

Bcl-2 homologous antagonist/killer is a protein that in humans isencoded by the BAK1 gene on chromosome 6. This protein localizes tomitochondria, and functions to induce apoptosis. It interacts with andaccelerates the opening of the mitochondrial voltage-dependent anionchannel, which leads to a loss in membrane potential and the release ofcytochrome c.

Human genes encoding proteins that belong to this family include: BAK1,BAX, BCL2, BCL2A1, BCL2L1, BCL2L2, BCL2L10, BCL2L13, BCL2L14, BOK, andMCL1.

2. Antiviral

Methods of the invention include using a programmable or targetablenuclease to specifically target viral nucleic acid for destruction. Anysuitable targeting nuclease can be used including, for example,zinc-finger nucleases (ZFNs), transcription activator-like effectornucleases (TALENs), clustered regularly interspaced short palindromicrepeat (CRISPR) nucleases, meganucleases, other endo- or exo-nucleases,or combinations thereof. See Schiffer, 2012, Targeted DNA mutagenesisfor the cure of chronic viral infections, J Virol 88(17):8920-8936,incorporated by reference.

CRISPR methodologies employ a nuclease, CRISPR-associated (Cas9), thatcomplexes with small RNAs as guides (gRNAs) to cleave DNA in asequence-specific manner upstream of the protospacer adjacent motif(PAM) in any genomic location. CRISPR may use separate guide RNAs knownas the crRNA and tracrRNA. These two separate RNAs have been combinedinto a single RNA to enable site-specific mammalian genome cuttingthrough the design of a short guide RNA. Cas9 and guide RNA (gRNA) maybe synthesized by known methods. Cas9/guide-RNA (gRNA) uses anon-specific DNA cleavage protein Cas9, and an RNA oligo to hybridize totarget and recruit the Cas9/gRNA complex. See Chang et al., 2013, Genomeediting with RNA-guided Cas9 nuclease in zebrafish embryos, Cell Res23:465-472; Hwang et al., 2013, Efficient genome editing in zebrafishusing a CRISPR-Cas system, Nat. Biotechnol 31:227-229; Xiao et al.,2013, Chromosomal deletions and inversions mediated by TALENS andCRISPR/Cas in zebrafish, Nucl Acids Res 1-11.

CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats) isfound in bacteria and is believed to protect the bacteria from phageinfection. It has recently been used as a means to alter gene expressionin eukaryotic DNA, but has not been proposed as an anti-viral therapy ormore broadly as a way to disrupt genomic material. Rather, it has beenused to introduce insertions or deletions as a way of increasing ordecreasing transcription in the DNA of a targeted cell or population ofcells. See for example, Horvath et al., Science (2010) 327:167-170;Terns et al., Current Opinion in Microbiology (2011) 14:321-327; Bhayaet al. Annu Rev Genet (2011) 45:273-297; Wiedenheft et al. Nature (2012)482:331-338); Jinek M et al. Science (2012) 337:816-821; Cong L et al.Science (2013) 339:819-823; Jinek M et al. (2013) eLife 2:e00471; Mali Pet al. (2013) Science 339:823-826; Qi LS et al. (2013) Cell152:1173-1183; Gilbert LA et al. (2013) Cell 154:442-451; Yang H et al.(2013) Cell 154:1370-1379; and Wang H et al. (2013) Cell 153:910-918).Additionally, in the co-pending U.S. Provisional Application 62/005,395it has been proposed as an anti-viral therapy or more broadly as a wayto disrupt genomic material.

In an aspect of the invention, the Cas9 endonuclease causes a doublestrand break in at least two locations in the genome. These two doublestrand breaks cause a fragment of the genome to be deleted. Even ifviral repair pathways anneal the two ends, there will still be adeletion in the genome. One or more deletions using the mechanism willincapacitate the viral genome. The result is that the host cell will befree of viral infection.

In embodiments of the invention, nucleases cleave the genome of thetarget virus. A nuclease is an enzyme capable of cleaving thephosphodiester bonds between the nucleotide subunits of nucleic acids.Endonucleases are enzymes that cleave the phosphodiester bond within apolynucleotide chain. Some, such as Deoxyribonuclease I, cut DNArelatively nonspecifically (without regard to sequence), while many,typically called restriction endonucleases or restriction enzymes,cleave only at very specific nucleotide sequences. In a preferredembodiment of the invention, the Cas9 nuclease is incorporated into thecompositions and methods of the invention, however, it should beappreciated that any nuclease may be utilized.

In preferred embodiments of the invention, the Cas9 nuclease is used tocleave the genome. The Cas9 nuclease is capable of creating a doublestrand break in the genome. The Cas9 nuclease has two functionaldomains: RuvC and HNH, each cutting a different strand. When both ofthese domains are active, the Cas9 causes double strand breaks in thegenome.

In some embodiments of the invention, insertions into the genome can bedesigned to cause incapacitation, or altered genomic expression.Additionally, insertions/deletions are also used to introduce apremature stop codon either by creating one at the double strand breakor by shifting the reading frame to create one downstream of the doublestrand break. Any of these outcomes of the NHEJ repair pathway can beleveraged to disrupt the target gene. The changes introduced by the useof the CRISPR/gRNA/Cas9 system are permanent to the genome.

In some embodiments of the invention, at least one insertion is causedby the CRISPR/gRNA/Cas9 complex. In a preferred embodiment, numerousinsertions are caused in the genome, thereby incapacitating the virus.In an aspect of the invention, the number of insertions lowers theprobability that the genome may be repaired.

In some embodiments of the invention, at least one deletion is caused bythe CRISPR/gRNA/Cas9 complex. In a preferred embodiment, numerousdeletions are caused in the genome, thereby incapacitating the virus. Inan aspect of the invention, the number of deletions lowers theprobability that the genome may be repaired. In a highly-preferredembodiment, the CRISPR/Cas9/gRNA system of the invention causessignificant genomic disruption, resulting in effective destruction ofthe viral genome, while leaving the host genome intact.

TALENs uses a nonspecific DNA-cleaving nuclease fused to a DNA-bindingdomain that can be to target essentially any sequence. For TALENtechnology, target sites are identified and expression vectors are made.Linearized expression vectors (e.g., by Notl) may be used as templatefor mRNA synthesis. A commercially available kit may be use such as themMESSAGE mMACHINE SP6 transcription kit from Life Technologies(Carlsbad, CA). See Joung & Sander, 2013, TALENs: a wideliy applicabletechnology for targeted genome editing, Nat Rev Mol Cell Bio 14:49-55.

TALENs and CRISPR methods provide one-to-one relationship to the targetsites, i.e. one unit of the tandem repeat in the TALE domain recognizesone nucleotide in the target site, and the crRNA, gRNA, or sgRNA ofCRISPR/Cas system hybridizes to the complementary sequence in the DNAtarget. Methods can include using a pair of TALENs or a Cas9 proteinwith one gRNA to generate double-strand breaks in the target. The breaksare then repaired via non-homologous end-joining or homologousrecombination (HR).

FIG. 8 shows ZFN being used to cut viral nucleic acid. Briefly, the ZFNmethod includes introducing into the infected host cell at least onevector (e.g., RNA molecule) encoding a targeted ZFN 305 and, optionally,at least one accessory polynucleotide. See, e.g., U.S. Pub. 2011/0023144to Weinstein, incorporated by reference The cell includes targetsequence 311. The cell is incubated to allow expression of the ZFN 305,wherein a double-stranded break 317 is introduced into the targetedchromosomal sequence 311 by the ZFN 305. In some embodiments, a donorpolynucleotide or exchange polynucleotide 321 is introduced. Swapping aportion of the viral nucleic acid with irrelevant sequence can fullyinterfere transcription or replication of the viral nucleic acid. TargetDNA 311 along with exchange polynucleotide 321 may be repaired by anerror-prone non-homologous end-joining DNA repair process or ahomology-directed DNA repair process.

Typically, a ZFN comprises a DNA binding domain (i.e., zinc finger) anda cleavage domain (i.e., nuclease) and this gene may be introduced asmRNA (e.g., 5′ capped, polyadenylated, or both). Zinc finger bindingdomains may be engineered to recognize and bind to any nucleic acidsequence of choice. See, e.g., Qu et al., 2013, Zinc-finger-nucleasesmediate specific and efficient excision of HIV-1 proviral DAN frominfected and latently infected human T cells, Nucl Ac Res41(16):7771-7782, incorporated by reference. An engineered zinc fingerbinding domain may have a novel binding specificity compared to anaturally-occurring zinc finger protein. Engineering methods include,but are not limited to, rational design and various types of selection.A zinc finger binding domain may be designed to recognize a target DNAsequence via zinc finger recognition regions (i.e., zinc fingers). Seefor example, U.S. Pat. Nos. 6,607,882; 6,534,261 and 6,453,242,incorporated by reference. Exemplary methods of selecting a zinc fingerrecognition region may include phage display and two-hybrid systems, andare disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523; 6,007,988;6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568, each of whichis incorporated by reference.

A ZFN also includes a cleavage domain. The cleavage domain portion ofthe ZFNs may be obtained from any suitable endonuclease or exonucleasesuch as restriction endonucleases and homing endonucleases. See, forexample, Belfort & Roberts, 1997, Homing endonucleases: keeping thehouse in order, Nucleic Acids Res 25(17):3379-3388. A cleavage domainmay be derived from an enzyme that requires dimerization for cleavageactivity. Two ZFNs may be required for cleavage, as each nucleasecomprises a monomer of the active enzyme dimer. Alternatively, a singleZFN may comprise both monomers to create an active enzyme dimer.Restriction endonucleases present may be capable of sequence-specificbinding and cleavage of DNA at or near the site of binding. Certainrestriction enzymes (e.g., Type IIS) cleave DNA at sites removed fromthe recognition site and have separable binding and cleavage domains.For example, the Type IIS enzyme Fokl, active as a dimer, catalyzesdouble-stranded cleavage of DNA, at 9 nucleotides from its recognitionsite on one strand and 13 nucleotides from its recognition site on theother. The Fokl enzyme used in a ZFN may be considered a cleavagemonomer. Thus, for targeted double-stranded cleavage using a Foklcleavage domain, two ZFNs, each comprising a Fokl cleavage monomer, maybe used to reconstitute an active enzyme dimer. See Wah, et al., 1998,Structure of Fokl has implications for DNA cleavage, PNAS95:10564-10569; U.S. Pat. Nos. 5,356,802; 5,436,150; 5,487,994; U.S.Pub. 2005/0064474; U.S. Pub. 2006/0188987; and U.S. Pub. 2008/0131962,each incorporated by reference.

Virus targeting using ZFN may include introducing at least one donorpolynucleotide comprising a sequence into the cell. A donorpolynucleotide preferably includes the sequence to be introduced flankedby an upstream and downstream sequence that share sequence similaritywith either side of the site of integration in the chromosome. Theupstream and downstream sequences in the donor polynucleotide areselected to promote recombination between the chromosomal sequence ofinterest and the donor polynucleotide. Typically, the donorpolynucleotide will be DNA. The donor polynucleotide may be a DNAplasmid, a bacterial artificial chromosome (BAC), a yeast artificialchromosome (YAC), a viral vector, a linear piece of DNA, a PCR fragment,a naked nucleic acid, and may employ a delivery vehicle such as aliposome. The sequence of the donor polynucleotide may include exons,introns, regulatory sequences, or combinations thereof. The doublestranded break is repaired via homologous recombination with the donorpolynucleotide such that the desired sequence is integrated into thechromosome. In the ZFN-mediated process for modifying a chromosomalsequence, a double stranded break introduced into the chromosomalsequence by the ZFN is repaired, via homologous recombination with theexchange polynucleotide, such that the sequence in the exchangepolynucleotide may be exchanged with a portion of the chromosomalsequence. The presence of the double stranded break facilitateshomologous recombination and repair of the break. The exchangepolynucleotide may be physically integrated or, alternatively, theexchange polynucleotide may be used as a template for repair of thebreak, resulting in the exchange of the sequence information in theexchange polynucleotide with the sequence information in that portion ofthe chromosomal sequence. Thus, a portion of the viral nucleic acid maybe converted to the sequence of the exchange polynucleotide. ZFN methodscan include using a vector to deliver a nucleic acid molecule encoding aZFN and, optionally, at least one exchange polynucleotide or at leastone donor polynucleotide to the infected cell.

Meganucleases are endodeoxyribonucleases characterized by a largerecognition site (double-stranded DNA sequences of 12 to 40 base pairs);as a result this site generally occurs only once in any given genome.For example, the 18-base pair sequence recognized by the I-SceImeganuclease would on average require a genome twenty times the size ofthe human genome to be found once by chance (although sequences with asingle mismatch occur about three times per human-sized genome).Meganucleases are therefore considered to be the most specific naturallyoccurring restriction enzymes. Meganucleases can be divided into fivefamilies based on sequence and structure motifs: LAGLIDADG, GIY-YIG,HNH, His-Cys box and PD-(D/E)XK. The most well studied family is that ofthe LAGLIDADG proteins, which have been found in all kingdoms of life,generally encoded within introns or inteins although freestandingmembers also exist. The sequence motif, LAGLIDADG, represents anessential element for enzymatic activity. Some proteins contained onlyone such motif, while others contained two; in both cases the motifswere followed by ˜75-200 amino acid residues having little to nosequence similarity with other family members. Crystal structuresillustrates mode of sequence specificity and cleavage mechanism for theLAGLIDADG family: (i) specificity contacts arise from the burial ofextended β-strands into the major groove of the DNA, with the DNAbinding saddle having a pitch and contour mimicking the helical twist ofthe DNA; (ii) full hydrogen bonding potential between the protein andDNA is never fully realized; (iii) cleavage to generate thecharacteristic 4-nt 3′-OH overhangs occurs across the minor groove,wherein the scissile phosphate bonds are brought closer to the proteincatalytic core by a distortion of the DNA in the central “4-base”region; (iv) cleavage occurs via a proposed two-metal mechanism,sometimes involving a unique “metal sharing” paradigm; (v) and finally,additional affinity and/or specificity contacts can arise from “adapted”scaffolds, in regions outside the core α/β fold. See Silva et al., 2011,Meganucleases and other tools for targeted genome engineering, Curr GeneTher 11(1):11-27, incorporated by reference.

In some embodiments of the invention, a template sequence is insertedinto the genome. In order to introduce nucleotide modifications togenomic DNA, a DNA repair template containing the desired sequence mustbe present during HDR. The DNA template is normally transfected into thecell along with the gRNA/Cas9. The length and binding position of eachhomology arm is dependent on the size of the change being introduced. Inthe presence of a suitable template, HDR can introduce specificnucleotide changes at the Cas9 induced double strand break.

Some embodiments of the invention may utilize modified version of anuclease. Modified versions of the Cas9 enzyme containing a singleinactive catalytic domain, either RuvC- or HNH-, are called ‘nickases’.With only one active nuclease domain, the Cas9 nickase cuts only onestrand of the target DNA, creating a single-strand break or ‘nick’.Similar to the inactive dCas9 (RuvC- and HNH-), a Cas9 nickase is stillable to bind DNA based on gRNA specificity, though nickases will onlycut one of the DNA strands. The majority of CRISPR plasmids are derivedfrom S. pyogenes and the RuvC domain can be inactivated by a D10Amutation and the HNH domain can be inactivated by an H840A mutation.

A single-strand break, or nick, is normally quickly repaired through theHDR pathway, using the intact complementary DNA strand as the template.However, two proximal, opposite strand nicks introduced by a Cas9nickase are treated as a double strand break, in what is often referredto as a ‘double nick’ or ‘dual nickase’ CRISPR system. A double-nickinduced double strain break can be repaired by either NHEJ or HDRdepending on the desired effect on the gene target. At these doublestrain breaks, insertions and deletions are caused by the CRISPR/Cas9complex. In an aspect of the invention, a deletion is caused bypositioning two double strand breaks proximate to one another, therebycausing a fragment of the genome to be deleted.

iii. Targeting Moiety

A nuclease may use the targeting specificity of a gRNA in order to act.As discussed below, guide RNAs or single guide RNAs are specificallydesigned to target a virus genome.

A CRISPR/Cas9 gene editing complex of the invention works optimally witha guide RNA that targets the viral genome. Guide RNA (gRNA) or singleguide RNA (sgRNA) leads the CRISPR/Cas9 complex to the viral genome inorder to cause viral genomic disruption. In an aspect of the invention,CRISPR/Cas9/gRNA complexes are designed to target specific viruseswithin a cell. It should be appreciated that any virus can be targetedusing the composition of the invention. Identification of specificregions of the virus genome aids in development and designing ofCRISPR/Cas9/gRNA complexes.

In an aspect of the invention, the CRISPR/Cas9/gRNA complexes aredesigned to target latent viruses within a cell. Once transfected withina cell, the CRISPR/Cas9/gRNA complexes cause repeated insertions ordeletions to render the genome incapacitated, or due to number ofinsertions or deletions, the probability of repair is significantlyreduced.

As an example, the Epstein-Barr virus (EBV), also called humanherpesvirus 4 (HHV-4) is inactivated in cells by a CRISPR/Cas9/gRNAcomplex of the invention. EBV is a virus of the herpes family, and isone of the most common viruses in humans. The virus is approximately 122nm to 180 nm in diameter and is composed of a double helix of DNAwrapped in a protein capsid. In this example, the Raji cell line servesas an appropriate in vitro model. The Raji cell line is the firstcontinuous human cell line from hematopoietic origin and cell linesproduce an unusual strain of Epstein-Barr virus while being one of themost extensively studied EBV models. To target the EBV genomes in theRaji cells, a CRISPR/Cas9 complex with specificity for EBV is needed.The design of EBV-targeting CRISPR/Cas9 plasmids consisting of a U6promoter driven chimeric guide RNA (sgRNA) and a ubiquitous promoterdriven Cas9 that were obtained from Addgene, Inc. Commercially availableguide RNAs and Cas9 nucleases may be used with the present invention. AnEGFP marker fused after the Cas9 protein allowed selection ofCas9-positive cells (FIG. 1A).

In an aspect of the invention, guide RNAs are designed, whether or notcommercially purchased, to target a specific viral genome. The viralgenome is identified and guide RNA to target selected portions of theviral genome are developed and incorporated into the composition of theinvention. In an aspect of the invention, a reference genome of aparticular strain of the virus is selected for guide RNA design.

For example, guide RNAs that target the EBV genome are a component ofthe system in the present example. In relation to EBV, for example, thereference genome from strain B95-8 was used as a design guide. Within agenome of interest, such as EBV, selected regions, or genes aretargeted. For example, six regions can be targeted with seven guide RNAdesigns for different genome editing purposes (FIG. 1C and Table S1).

TABLE S1 Guide RNA target sequences Guide RNA SequencesgEBV1 GCCCTGGACCAACCCGGCCC (SEQ ID NO: 1)sgEBV2 GGCCGCTGCCCCGCTCCGGG (SEQ ID NO: 2)sgEBB3 GGAAGACAATGTGCCGCCA (SEQ ID NO: 3)sgEBV4 TCTGGACCAGAAGGCTCCGG (SEQ ID NO: 4)sgEBV5 GCTGCCGCGGAGGGTGATGA (SEQ ID NO: 5)sgEBV6 GGTGGCCCACCGGGTCCGCT (SEQ ID NO: 6)sgEBV7 GTCCTCGAGGGGGCCGTCGC (SEQ ID NO: 7)

In relation to EBV, EBNA1 is the only nuclear Epstein-Barr virus (EBV)protein expressed in both latent and lytic modes of infection. WhileEBNA1 is known to play several important roles in latent infection,EBNA1 is crucial for many EBV functions including gene regulation andlatent genome replication. Therefore, guide RNAs sgEBV4 and sgEBV5 wereselected to target both ends of the EBNA1 coding region in order toexcise this whole region of the genome. These “structural” targetsenable systematic digestion of the EBV genome into smaller pieces.EBNA3C and LMP1 are essential for host cell transformation, and guideRNAs sgEBV3 and sgEBV7 were designed to target the 5′ exons of these twoproteins respectively.

iv. Introduce to Cell

Methods of the invention include introducing into a cell a nuclease anda sequence-specific targeting moiety. The nuclease is targeted to viralnucleic acid by means of the sequence-specific targeting moiety where itthen cleaves the viral nucleic acid without interfering with a hostgenome. Any suitable method can be used to deliver the nuclease to theinfected cell or tissue. For example, the nuclease or the gene encodingthe nuclease may be delivered by injection, orally, or by hydrodynamicdelivery. The nuclease or the gene encoding the nuclease may bedelivered to systematic circulation or may be delivered or otherwiselocalized to a specific tissue type.

Some viral infections affect only a small number of cells, and it may bepreferable to employ a targeted delivery approach. It has been estimatedthat HSV, for example, latently infects on the order of only 20,000neurons. For this and other viral infections, it may be important tohave a treatment that is targeted only to the cells of interest.Increasing the cell affinity and specificity can greatly improvetherapeutic delivery efficiency.

The nuclease or gene encoding the nuclease may be modified or programmedto be active under only certain conditions such as by using atissue-specific promoter so that the encoded nuclease is preferentiallyor only transcribed in certain tissue types.

In some embodiments, specific CRISPR/Cas9/gRNA complexes are introducedinto a cell. A guide RNA is designed to target at least one category ofsequences of the viral genome. In addition to latent infections thisinvention can also be used to control actively replicating viruses bytargeting the viral genome before it is packaged or after it is ejected.

Prepackaged GFP-Cas9-adenovirus is available from Vector Biolabs(Philadelphia, Pa.). Various targeting gRNA sequences, such as sequencesthat target EBV can be packaged to adenovirus lines. The gRNA sequencescan be housed together with the CRISPR/Cas9 complex or separately.

In some embodiments, a cocktail of guide RNAs may be introduced into acell. The guide RNAs are designed to target numerous categories ofsequences of the viral genome. By targeting several areas along thegenome, the double strand break at multiple locations fragments thegenome, lowering the possibility of repair. Even with repair mechanisms,the large deletions render the virus incapacitated.

In some embodiments, several guide RNAs are added to create a cocktailto target different categories of sequences. For example, two, five,seven or eleven guide RNAs may be present in a CRISPR cocktail targetingthree different categories of sequences. However, any number of gRNAsmay be introduced into a cocktail to target categories of sequences. Inpreferred embodiments, the categories of sequences are important forgenome structure, host cell transformation, and infection latency,respectively.

In some aspects of the invention, in vitro experiments allow for thedetermination of the most essential targets within a viral genome. Forexample, to understand the most essential targets for effectiveincapacitation of a genome, subsets of guide RNAs are transfected intomodel cells. Assays can determine which guide RNAs or which cocktail isthe most effective at targeting essential categories of sequences.

These agents can be delivered either as part of a viral vector (examplesfurther described below), or as naked as DNA or RNA. Naked nucleic acidscan be modified to avoid degradation.

Another possibility is to deliver the protein product itself eitherfused to a signaling molecule or packaged into a vesicle with signalingmolecules on surface, or packed into a nanoparticle, vesicle, orattached to a colloid. Examples of this method of delivery have beenpreviously explored in cancer but not applied to local delivery againstlatent viral infections. (See Alexis et al, “Nanoparticle Technologiesfor Cancer Therapy” in Drug Delivery, Handbook of ExperimentalPharmacology 197, 2010.) Other delivery methods are described in detailbelow. For HSV and other viruses which are highly localized in terms ofwhich cells and tissues they infect, these therapies might be deliveredas a local injection or as a cream.

In other embodiments of the invention, physical approaches can be usedto ablate cells that have latent infection, taking advantage of the factthat these are localized in diseases such as HSV. One approach is toimage infected cells (for example, using fluorescent markers againstviral protein, or against viral genome, or fluorescence proteins inducedby viral latency promoters) and then use heat, light, or radio frequencyradiation to ablate those cells. Direct contrast agents can also be usedtowards infected cells. Instead of fluorescence molecules, semiconductoror metallic nanoparticles, colloids, or other structures that interactstrongly with light or radio frequency can be used. These can be appliedlocally with a cream or injections. These substances can potentiallytake advantage of a cooperative effect, such that infected cells attractmultiple particles, thereby having the highest effect. Similarapproaches have been used in cancer treatment (See for example Jain etal “Gold nanoparticles as novel agents for cancer therapy,” Br J RadiolFeb 2012 85(1010):101-113). The present invention applies thesetechniques to treatment of latent viral infection.

In another embodiment, labeled and infected cells can be excised usingmicrosurgery tools such as a fiber optic endoscope, which allows imagingand delivery of radiation in a highly localized manner, with single cellresolution. (See Barretto R P and Schnitzer M J. “In Vivo OpticalMicroendoscopy for Imaging Cells Lying Deep within Live Tissue.” ColdSpring Harb Protoc. 2012(10) and Llewellyn M E, Barretto R P J, Delp S L& Schnitzer M J. (2008) Minimally invasive high-speed imaging ofsarcomere contractile dynamics in mice and humans. Nature. 454 784-788).

For example, in the case of the EBV genome targeting, seven guide RNAsin the CRISPR cocktail targeted three different categories of sequenceswhich are identified as being important for EBV genome structure, hostcell transformation, and infection latency, respectively. To understandthe most essential targets for effective EBV treatment, Raji cells weretransfected with subsets of guide RNAs. Although sgEBV4/5 reduced theEBV genome by 85%, they could not suppress cell proliferation aseffectively as the full cocktail (FIG. 3A). Guide RNAs targeting thestructural sequences (sgEBV1/2/6) could stop cell proliferationcompletely, despite not eliminating the full EBV load (26% decrease).Given the high efficiency of genome editing and the proliferation arrest(FIG. 2), it was suspect that the residual EBV genome signature insgEBV1/2/6 was not due to intact genomes but to free-floating DNA thathas been digested out of the EBV genome, i.e. as a false positive.

Once CRISPR/Cas9/gRNA complexes are constructed, the complexes areintroduced into a cell. It should be appreciated that complexes can beintroduced into cells in an in vitro model or an in vivo model. In anaspect of the invention, CRISPR/Cas9/gRNA complexes are designed to notleave intact genomes of a virus after transfection and complexes aredesigned for efficient transfection.

Aspects of the invention allow for CRISPR/Cas9/gRNA to be transfectedinto cells by various methods, including viral vectors and non-viralvectors. Viral vectors may include retroviruses, lentiviruses,adenoviruses, and adeno-associated viruses. It should be appreciatedthat any viral vector may be incorporated into the present invention toeffectuate delivery of the CRISPR/Cas9/gRNA complex into a cell. Someviral vectors may be more effective than others, depending on theCRISPR/Cas9/gRNA complex designed for digestion or incapacitation. In anaspect of the invention, the vectors contain essential components suchas origin of replication, which is necessary for the replication andmaintenance of the vector in the host cell.

In an aspect of the invention, viral vectors are used as deliveryvectors to deliver the complexes into a cell. Use of viral vectors asdelivery vectors are known in the art. See for example U.S. Pub.2009/0017543 to Wilkes et al., the contents of which are incorporated byreference.

A retrovirus is a single-stranded RNA virus that stores its nucleic acidin the form of an mRNA genome (including the 5′ cap and 3′ PolyA tail)and targets a host cell as an obligate parasite. In some methods in theart, retroviruses have been used to introduce nucleic acids into a cell.Once inside the host cell cytoplasm the virus uses its own reversetranscriptase enzyme to produce DNA from its RNA genome, the reverse ofthe usual pattern, thus retro (backwards). This new DNA is thenincorporated into the host cell genome by an integrase enzyme, at whichpoint the retroviral DNA is referred to as a provirus. For example, therecombinant retroviruses such as the Moloney murine leukemia virus havethe ability to integrate into the host genome in a stable fashion. Theycontain a reverse transcriptase that allows integration into the hostgenome. Retroviral vectors can either be replication-competent orreplication-defective. In some embodiments of the invention,retroviruses are incorporated to effectuate transfection into a cell,however the CRISPR/Cas9/gRNA complexes are designed to target the viralgenome.

In some embodiments of the invention, lentiviruses, which are a subclassof retroviruses, are used as viral vectors. Lentiviruses can be adaptedas delivery vehicles (vectors) given their ability to integrate into thegenome of non-dividing cells, which is the unique feature oflentiviruses as other retroviruses can infect only dividing cells. Theviral genome in the form of RNA is reverse-transcribed when the virusenters the cell to produce DNA, which is then inserted into the genomeat a random position by the viral integrase enzyme. The vector, nowcalled a provirus, remains in the genome and is passed on to the progenyof the cell when it divides.

As opposed to lentiviruses, adenoviral DNA does not integrate into thegenome and is not replicated during cell division. Adenovirus and therelated AAV would be potential approaches as delivery vectors since theydo not integrate into the host's genome. In some aspects of theinvention, only the viral genome to be targeted is effected by theCRISPR/Cas9/gRNA complexes, and not the host's cells. Adeno-associatedvirus (AAV) is a small virus that infects humans and some other primatespecies. AAV can infect both dividing and non-dividing cells and mayincorporate its genome into that of the host cell. For example, becauseof its potential use as a gene therapy vector, researchers have createdan altered AAV called self-complementary adeno-associated virus (scAAV).Whereas AAV packages a single strand of DNA and requires the process ofsecond-strand synthesis, scAAV packages both strands which annealtogether to form double stranded DNA. By skipping second strandsynthesis scAAV allows for rapid expression in the cell. Otherwise,scAAV carries many characteristics of its AAV counterpart. Methods ofthe invention may incorporate herpesvirus, poxvirus, alphavirus, orvaccinia virus as a means of delivery vectors.

In certain embodiments of the invention, non-viral vectors may be usedto effectuate transfection. Methods of non-viral delivery of nucleicacids include lipofection, nucleofection, microinjection, biolistics,virosomes, liposomes, micelles, immunoliposomes, polycation orlipid:nucleic acid conjugates, naked DNA, artificial virions, andagent-enhanced uptake of DNA. Lipofection is described in e.g., U.S.Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagentsare sold commercially (e.g., Transfectam and Lipofectin). Cationic andneutral lipids that are suitable for efficient receptor-recognitionlipofection of polynucleotides include those described in U.S. Pat.7,166,298 to Jessee or U.S. Pat. 6,890,554 to Jesse, the contents ofeach of which are incorporated by reference. Delivery can be to cells(e.g. in vitro or ex vivo administration) or target tissues (e.g. invivo administration).

Synthetic vectors are typically based on cationic lipids or polymerswhich can complex with negatively charged nucleic acids to formparticles with a diameter in the order of 100 nm. The complex protectsnucleic acid from degradation by nuclease. Moreover, cellular and localdelivery strategies have to deal with the need for internalization,release, and distribution in the proper subcellular compartment.Systemic delivery strategies encounter additional hurdles, for example,strong interaction of cationic delivery vehicles with blood components,uptake by the reticuloendothelial system, kidney filtration, toxicityand targeting ability of the carriers to the cells of interest.Modifying the surfaces of the cationic non-virals can minimize theirinteraction with blood components, reduce reticuloendothelial systemuptake, decrease their toxicity and increase their binding affinity withthe target cells. Binding of plasma proteins (also termed opsonization)is the primary mechanism for RES to recognize the circulatingnanoparticles. For example, macrophages, such as the Kupffer cells inthe liver, recognize the opsonized nanoparticles via the scavengerreceptor.

In some embodiments of the invention, non-viral vectors are modified toeffectuate targeted delivery and transfection. PEGylation (i.e.modifying the surface with polyethyleneglycol) is the predominant methodused to reduce the opsonization and aggregation of non-viral vectors andminimize the clearance by reticuloendothelial system, leading to aprolonged circulation lifetime after intravenous (i.v.) administration.PEGylated nanoparticles are therefore often referred as “stealth”nanoparticles. The nanoparticles that are not rapidly cleared from thecirculation will have a chance to encounter infected cells.

However, PEG on the surface can decrease the uptake by target cells andreduce the biological activity. Therefore, to attach targeting ligand tothe distal end of the PEGylated component is necessary; the ligand isprojected beyond the PEG “shield” to allow binding to receptors on thetarget cell surface. When cationic liposome is used as gene carrier, theapplication of neutral helper lipid is helpful for the release ofnucleic acid, besides promoting hexagonal phase formation to enableendosomal escape. In some embodiments of the invention, neutral oranionic liposomes are developed for systemic delivery of nucleic acidsand obtaining therapeutic effect in experimental animal model. Designingand synthesizing novel cationic lipids and polymers, and covalently ornoncovalently binding gene with peptides, targeting ligands, polymers,or environmentally sensitive moieties also attract many attentions forresolving the problems encountered by non-viral vectors. The applicationof inorganic nanoparticles (for example, metallic nanoparticles, ironoxide, calcium phosphate, magnesium phosphate, manganese phosphate,double hydroxides, carbon nanotubes, and quantum dots) in deliveryvectors can be prepared and surface-functionalized in many differentways.

In some embodiments of the invention, targeted controlled-releasesystems responding to the unique environments of tissues and externalstimuli are utilized. Gold nanorods have strong absorption bands in thenear-infrared region, and the absorbed light energy is then convertedinto heat by gold nanorods, the so-called ‘photothermal effect’. Becausethe near-infrared light can penetrate deeply into tissues, the surfaceof gold nanorod could be modified with nucleic acids for controlledrelease. When the modified gold nanorods are irradiated by near-infraredlight, nucleic acids are released due to thermo-denaturation induced bythe photothermal effect. The amount of nucleic acids released isdependent upon the power and exposure time of light irradiation.

In some embodiments of the invention, liposomes are used to effectuatetransfection into a cell or tissue. A “liposome” as used herein refersto a small, spherical vesicle composed of lipids, particularlyvesicle-forming lipids capable of spontaneously arranging into lipidbilayer structures in water with its hydrophobic moiety in contact withthe interior, hydrophobic region of the bilayer membrane, and its headgroup moiety oriented toward the exterior, polar surface of themembrane. Vesicle-forming lipids have typically two hydrocarbon chains,particularly acyl chains, and a head group, either polar or nonpolar.Vesicle-forming lipids are either composed of naturally-occurring lipidsor of synthetic origin, including the phospholipids, such asphosphatidylcholine, phosphatidylethanolamine, phosphatidic acid,phosphatidylinositol, and sphingomyelin, where the two hydrocarbonchains are typically between about 14-22 carbon atoms in length, andhave varying degrees of unsaturation. The above-described lipids andphospholipids whose acyl chains have varying degrees of saturation canbe obtained commercially or prepared according to published methods.Other suitable lipids for use in the composition of the presentinvention include glycolipids and sterols such as cholesterol and itsvarious analogs which can also be used in the liposomes.

Similar to a liposome, a micelle is a small spherical vesical composedof lipids, but is arranged as a lipid monolayer, with the hydrophilichead regions of the lipid molecules in contact with surrounding solvent,sequestering the hydrophobic single-tail regions in the center of themicelle. This phase is caused by the packing behavior of single-taillipids in a bilayer.

The pharmacology of a liposomal formulation of nucleic acid is largelydetermined by the extent to which the nucleic acid is encapsulatedinside the liposome bilayer. Encapsulated nucleic acid is protected fromnuclease degradation, while those merely associated with the surface ofthe liposome is not protected. Encapsulated nucleic acid shares theextended circulation lifetime and biodistribution of the intactliposome, while those that are surface associated adopt the pharmacologyof naked nucleic acid once they disassociate from the liposome.

In some embodiments, the complexes of the invention are encapsulated ina liposome. Unlike small molecule drugs, nucleic acids cannot crossintact lipid bilayers, predominantly due to the large size andhydrophilic nature of the nucleic acid. Therefore, nucleic acids may beentrapped within liposomes with conventional passive loadingtechnologies, such as ethanol drop method (as in SALP), reverse-phaseevaporation method, and ethanol dilution method (as in SNALP).

In some embodiments, linear polyethylenimine (L-PEI) is used as anon-viral vector due to its versatility and comparatively hightransfection efficiency. L-PEI has been used to efficiently delivergenes in vivo into a wide range of organs such as lung, brain, pancreas,retina, bladder as well as tumor. L-PEI is able to efficiently condense,stabilize and deliver nucleic acids in vitro and in vivo.

Low-intensity ultrasound in combination with microbubbles has recentlyacquired much attention as a safe method of gene delivery. Ultrasoundshows tissue-permeabilizing effect. It is non-invasive andsite-specific, and could make it possible to destroy tumor cells aftersystemic delivery, while leave nontargeted organs unaffected.Ultrasound-mediated microbubbles destruction has been proposed as aninnovative method for noninvasive delivering of drugs and nucleic acidsto different tissues. Microbubbles are used to carry a drug or geneuntil a specific area of interest is reached, and then ultrasound isused to burst the microbubbles, causing site-specific delivery of thebioactive materials. Furthermore, the ability of albumin-coatedmicrobubbles to adhere to vascular regions with glycocalix damage orendothelial dysfunction is another possible mechanism to deliver drugseven in the absence of ultrasound. See Tsutsui et al., 2004, The use ofmicrobubbles to target drug delivery, Cardiovasc Ultrasound 2:23, thecontents of which are incorporated by reference. In ultrasound-triggereddrug delivery, tissue-permeabilizing effect can be potentiated usingultrasound contrast agents, gas-filled microbubbles. The use ofmicrobubbles for delivery of nucleic acids is based on the hypothesisthat destruction of DNA-loaded microbubbles by a focused ultrasound beamduring their microvascular transit through the target area will resultin localized transduction upon disruption of the microbubble shell whilesparing non-targeted areas.

Besides ultrasound-mediated delivery, magnetic targeting delivery couldbe used for delivery. Magnetic nanoparticles are usually entrapped ingene vectors for imaging the delivery of nucleic acid. Nucleic acidcarriers can be responsive to both ultrasound and magnetic fields, i.e.,magnetic and acoustically active lipospheres (MAALs). The basic premiseis that therapeutic agents are attached to, or encapsulated within, amagnetic micro- or nanoparticle. These particles may have magnetic coreswith a polymer or metal coating which can be functionalized, or mayconsist of porous polymers that contain magnetic nanoparticlesprecipitated within the pores. By functionalizing the polymer or metalcoating it is possible to attach, for example, cytotoxic drugs fortargeted chemotherapy or therapeutic DNA to correct a genetic defect.Once attached, the particle/therapeutic agent complex is injected intothe bloodstream, often using a catheter to position the injection sitenear the target. Magnetic fields, generally from high-field,high-gradient, rare earth magnets are focused over the target site andthe forces on the particles as they enter the field allow them to becaptured and extravasated at the target.

Synthetic cationic polymer-based nanoparticles (˜100 nm diameter) havebeen developed that offer enhanced transfection efficiency combined withreduced cytotoxicity, as compared to traditional liposomes. Theincorporation of distinct layers composed of lipid molecules withvarying physical and chemical characteristics into the polymernanoparticle formulation resulted in improved efficiency through betterfusion with cell membrane and entry into the cell, enhanced release ofmolecules inside the cell, and reduced intracellular degradation ofnanoparticle complexes.

In some embodiments, the complexes are conjugated to nano-systems forsystemic therapy, such as liposomes, albumin-based particles, PEGylatedproteins, biodegradable polymer-drug composites, polymeric micelles,dendrimers, among others. See Davis et al., 2008, Nanotherapeuticparticles: an emerging treatment modality for cancer, Nat Rev DrugDiscov. 7(9):771-782, incorporated by reference. Long circulatingmacromolecular carriers such as liposomes, can exploit the enhancedpermeability and retention effect for preferential extravasation fromtumor vessels. In certain embodiments, the complexes of the inventionare conjugated to or encapsulated into a liposome or polymerosome fordelivery to a cell. For example, liposomal anthracyclines have achievedhighly efficient encapsulation, and include versions with greatlyprolonged circulation such as liposomal daunorubicin and pegylatedliposomal doxorubicin. See Krishna et al., Carboxymethylcellulose-sodiumbased transdermal drug delivery system for propranolol, J PharmPharmacol. 1996 April; 48(4):367-70.

Liposomal delivery systems provide stable formulation, provide improvedpharmacokinetics, and a degree of ‘passive’ or ‘physiological’ targetingto tissues. Encapsulation of hydrophilic and hydrophobic materials, suchas potential chemotherapy agents, are known. See for example U.S. Pat.No. 5,466,468 to Schneider, which discloses parenterally administrableliposome formulation comprising synthetic lipids; U.S. Pat. No.5,580,571, to Hostetler et al. which discloses nucleoside analoguesconjugated to phospholipids; U.S. Pat. No. 5,626,869 to Nyqvist, whichdiscloses pharmaceutical compositions wherein the pharmaceuticallyactive compound is heparin or a fragment thereof contained in a definedlipid system comprising at least one amphiphatic and polar lipidcomponent and at least one nonpolar lipid component.

Liposomes and polymerosomes can contain a plurality of solutions andcompounds. In certain embodiments, the complexes of the invention arecoupled to or encapsulated in polymersomes. As a class of artificialvesicles, polymersomes are tiny hollow spheres that enclose a solution,made using amphiphilic synthetic block copolymers to form the vesiclemembrane. Common polymersomes contain an aqueous solution in their coreand are useful for encapsulating and protecting sensitive molecules,such as drugs, enzymes, other proteins and peptides, and DNA and RNAfragments. The polymersome membrane provides a physical barrier thatisolates the encapsulated material from external materials, such asthose found in biological systems. Polymerosomes can be generated fromdouble emulsions by known techniques, see Lorenceau et al., 2005,Generation of Polymerosomes from Double-Emulsions, Langmuir21(20):9183-6, incorporated by reference.

Some embodiments of the invention provide for a gene gun or a biolisticparticle delivery system. A gene gun is a device for injecting cellswith genetic information, where the payload may be an elemental particleof a heavy metal coated with plasmid DNA. This technique may also bereferred to as bioballistics or biolistics. Gene guns have also beenused to deliver DNA vaccines. The gene gun is able to transfect cellswith a wide variety of organic and non-organic species, such as DNAplasmids, fluorescent proteins, dyes, etc.

Aspects of the invention provide for numerous uses of delivery vectors.Selection of the delivery vector is based upon the cell or tissuetargeted and the specific makeup of the CRISPR/Cas9/gRNA. For example,in the EBV example discussed above, since lymphocytes are known forbeing resistant to lipofection, nucleofection (a combination ofelectrical parameters generated by a device called Nucleofector, withcell-type specific reagents to transfer a substrate directly into thecell nucleus and the cytoplasm) was necessitated for DNA delivery intothe Raji cells. The Lonza pmax promoter drives Cas9 expression as itoffered strong expression within Raji cells. 24 hours afternucleofection, obvious EGFP signals were observed from a smallproportion of cells through fluorescent microscopy. The EGFP-positivecell population decreased dramatically, however, <10% transfectionefficiency 48 hours after nucleofection was measured (FIG. 1B). A CRISPRplasmid that included the EBV origin of replication sequence, oriPyielded a transfection efficiency >60% (FIG. 1B).

Aspects of the invention utilize the CRISPR/Cas9/gRNA complexes for thetargeted delivery. Common known pathways include transdermal,transmucal, nasal, ocular and pulmonary routes. Drug delivery systemsmay include liposomes, proliposomes, microspheres, gels, prodrugs,cyclodextrins, etc. Aspects of the invention utilize nanoparticlescomposed of biodegradable polymers to be transferred into an aerosol fortargeting of specific sites or cell populations in the lung, providingfor the release of the drug in a predetermined manner and degradationwithin an acceptable period of time. Controlled-release technology(CRT), such as transdermal and transmucosal controlled-release deliverysystems, nasal and buccal aerosol sprays, drug-impregnated lozenges,encapsulated cells, oral soft gels, iontophoretic devices to administerdrugs through skin, and a variety of programmable, implanteddrug-delivery devices are used in conjunction with the complexes of theinvention of accomplishing targeted and controlled delivery.

v. Digest Nucleic Acid

Once inside the cell, the CRISPR/Cas9/gRNA complexes target nucleicacid. In an aspect of the invention, the complexes are targeted to viralgenomes. In addition to latent infections this invention can also beused to control actively replicating viruses by targeting the viralgenome before it is packaged or after it is ejected. In someembodiments, methods and compositions of the invention use a nucleasesuch as Cas9 to target latent viral genomes, thereby reducing thechances of proliferation. The nuclease may form a complex with a gRNA(e.g., crRNA+tracrRNA or sgRNA). The complex cuts the viral nucleic acidin a targeted fashion to incapacitate the viral genome. As discussedabove, the Cas9 endonuclease causes a double strand break in the viralgenome. By targeted several locations along the viral genome and causingnot a single strand break, but a double strand break, the genome iseffectively cut a several locations along the genome. In a preferredembodiment, the double strand breaks are designed so that smalldeletions are caused, or small fragments are removed from the genome sothat even if natural repair mechanisms join the genome together, thegenome is render incapacitated.

After introduction into a cell, the CRISPR/Cas9/gRNA complexes act onthe viral genome, genes, transcripts, or other viral nucleic acid. Thedouble-strand DNA breaks generated by CRISPR are repaired with smalldeletions. These deletions will disrupt the protein coding and hencecreate knockout effects.

The nuclease, or a gene encoding the nuclease, may be delivered into aninfected cell by transfection. For example, the infected cell can betransfected with DNA that encodes Cas9 and gRNA (on a single piece orseparate pieces). The gRNAs are designed to localize the Cas9endonuclease at one or several locations along the viral genome. TheCas9 endonuclease causes double strand breaks in the genome, causingsmall fragments to be deleted from the viral genome. Even with repairmechanisms, the deletions render the viral genome incapacitated.

Engineered viral particles with higher cell affinity (e.g. RGD knob) andspecificity could greatly improve delivery efficiency. Delivery ofcircular instead of linear DNA may also be beneficial since the circularDNA can replicate as episomes with replication origins.

Aspects of the invention utilize the CRISPR/Cas9/gRNA complexes for thetargeted delivery. Common known pathways include transdermal,transmucal, nasal, ocular and pulmonary routes. Drug delivery systemsmay include liposomes, proliposomes, micelles, microspheres, gels,prodrugs, cyclodextrins, etc. Aspects of the invention utilizenanoparticles composed of biodegradable polymers to be transferred intoan aerosol for targeting of specific sites or cell populations in thelung, providing for the release of the drug in a predetermined mannerand degradation within an acceptable period of time. Controlled-releasetechnology (CRT), such as transdermal and transmucosalcontrolled-release delivery systems, nasal and buccal aerosol sprays,drug-impregnated lozenges, encapsulated cells, oral soft gels,iontophoretic devices to administer drugs through skin, and a variety ofprogrammable, implanted drug-delivery devices are used in conjunctionwith the complexes of the invention of accomplishing targeted andcontrolled delivery.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

Equivalents

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

EXAMPLES Example 1 Targeting EBV

Burkitt's lymphoma cell lines Raji, Namalwa, and DG-75 were obtainedfrom ATCC and cultured in RPMI 1640 supplemented with 10% FBS and PSA,following ATCC recommendation. Human primary lung fibroblast IMR-90 wasobtained from Coriell and cultured in Advanced DMEM/F-12 supplementedwith 10% FBS and PSA.

Plasmids consisting of a U6 promoter driven chimeric guide RNA (sgRNA)and a ubiquitous promoter driven Cas9 were obtained from addgene, asdescribed by Cong L et al. (2013) Multiplex Genome Engineering UsingCRISPR/Cas Systems. Science 339:819-823. An EGFP marker fused after theCas9 protein allowed selection of Cas9-positive cells (FIG. 1A). Weadapted a modified chimeric guide RNA design for more efficient Pol-IIItranscription and more stable stem-loop structure (Chen B et al. (2013)Dynamic Imaging of Genomic Loci in Living Human Cells by an OptimizedCRISPR/Cas System. Cell 155:1479-1491).

We obtained pX458 from Addgene, Inc. A modified CMV promoter with asynthetic intron (pmax) was PCR amplified from Lonza control plasmidpmax-GFP. A modified guide RNA sgRNA(F+E) was ordered from IDT. EBVreplication origin oriP was PCR amplified from B95-8 transformedlymphoblastoid cell line GM12891. We used standard cloning protocols toclone pmax, sgRNA(F+E) and oriP to pX458, to replace the original CAGpromoter, sgRNA and fl origin. We designed EBV sgRNA based on the B95-8reference, and ordered DNA oligos from IDT. The original sgRNA placeholder in pX458 serves as the negative control.

Lymphocytes are known for being resistant to lipofection, and thereforewe used nucleofection for DNA delivery into Raji cells. We chose theLonza pmax promoter to drive Cas9 expression as it offered strongexpression within Raji cells. We used the Lonza Nucleofector II for DNAdelivery. 5 million Raji or DG-75 cells were transfected with 5 ugplasmids in each 100-ul reaction. Cell line Kit V and program M-013 wereused following Lonza recommendation. For IMR-90, 1 million cells weretransfected with 5 ug plasmids in 100 ul Solution V, with program T-030or X-005. 24 hours after nucleofection, we observed obvious EGFP signalsfrom a small proportion of cells through fluorescent microscopy. TheEGFP-positive cell population decreased dramatically after that,however, and we measured <10% transfection efficiency 48 hours afternucleofection (FIG. 1B). We attributed this transfection efficiencydecrease to the plasmid dilution with cell division. To activelymaintain the plasmid level within the host cells, we redesigned theCRISPR plasmid to include the EBV origin of replication sequence, oriP.With active plasmid replication inside the cells, the transfectionefficiency rose to >60% (FIG. 1B).

To design guide RNA targeting the EBV genome, we relied on the EBVreference genome from strain B95-8. We targeted six regions with sevenguide RNA designs for different genome editing purposes (FIG. 1C andTable S1). EBNA1 is crucial for many EBV functions including generegulation and latent genome replication. We targeted guide RNA sgEBV4and sgEBV5 to both ends of the EBNA1 coding region in order to excisethis whole region of the genome. Guide RNAs sgEBV1, 2 and 6 fall inrepeat regions, so that the success rate of at least one CRISPR cut ismultiplied. These “structural” targets enable systematic digestion ofthe EBV genome into smaller pieces. EBNA3C and LMP1 are essential forhost cell transformation, and we designed guide RNAs sgEBV3 and sgEBV7to target the 5′ exons of these two proteins respectively.

EBV Genome Editing. The double-strand DNA breaks generated by CRISPR arerepaired with small deletions. These deletions will disrupt the proteincoding and hence create knockout effects. SURVEYOR assays confirmedefficient editing of individual sites (FIG. 5). Beyond the independentsmall deletions induced by each guide RNA, large deletions betweentargeting sites can systematically destroy the EBV genome. Guide RNAsgEBV2 targets a region with twelve 125-bp repeat units (FIG. 2A). PCRamplicon of the whole repeat region gave a ˜1.8-kb band (FIG. 2B). After5 or 7 days of sgEBV2 transfection, we obtained ˜0.4-kb bands from thesame PCR amplification (FIG. 2B). The ˜1.4-kb deletion is the expectedproduct of repair ligation between cuts in the first and the last repeatunit (FIG. 2A).

DNA sequences flanking sgRNA targets were PCR amplified with Phusion DNApolymerase. SURVEYOR assays were performed following manufacturer'sinstruction. DNA amplicons with large deletions were TOPO cloned andsingle colonies were used for Sanger sequencing. EBV load was measuredwith Taqman digital PCR on Fluidigm BioMark. A Taqman assay targeting aconserved human locus was used for human DNA normalization. 1 ng ofsingle-cell whole-genome amplification products from Fluidigm Cl wereused for EBV quantitative PCR.

We further demonstrated that it is possible to delete regions betweenunique targets (FIG. 2C). Six days after sgEBV4-5 transfection, PCRamplification of the whole flanking region (with primers EBV4F and 5R)returned a shorter amplicon, together with a much fainter band of theexpected 2 kb (FIG. 2D). Sanger sequencing of amplicon clones confirmedthe direct connection of the two expected cutting sites (FIG. 2F). Asimilar experiment with sgEBV3-5 also returned an even larger deletion,from EBNA3C to EBNA1 (FIG. 2D-E).

Cell Proliferation Arrest With EBV Genome Destruction. Two days afterCRISPR transfection, we flow sorted EGFP-positive cells for furtherculture and counted the live cells daily. As expected, cells treatedwith Cas9 plasmids which lacked oriP or sgEBV lost EGFP expressionwithin a few days and proliferated with a rate similar rate to theuntreated control group (FIG. 3A). Plasmids with Cas9-oriP and ascrambled guide RNA maintained EGFP expression after 8 days, but did notreduce the cell proliferation rate. Treatment with the mixed cocktailsgEBV1-7 resulted in no measurable cell proliferation and the total cellcount either remained constant or decreased (FIG. 3A). Flow cytometryscattering signals clearly revealed alterations in the cell morphologyafter sgEBV1-7 treatment, as the majority of the cells shrank in sizewith increasing granulation (FIG. 3B-D, population P4 to P3 shift).Cells in population P3 also demonstrated compromised membranepermeability by DAPI staining (FIG. 3E-G). To rule out the possibilityof CRISPR cytotoxicity, especially with multiple guide RNAs, weperformed the same treatment on two other samples: the EBV-negativeBurkitt's lymphoma cell line DG-75 (FIG. 6) and primary human lungfibroblast IMR90 (FIG. 7). Eight and nine days after transfection thecell proliferation rates did not change from the untreated controlgroups, suggesting neglectable cytotoxicity.

Previous studies have attributed the EBV tumorigenic ability to itsinterruption of host cell apoptosis (Ruf IK et al. (1999) Epstein-BarrVirus Regulates c-MYC, Apoptosis, and Tumorigenicity in BurkittLymphoma. Molecular and Cellular Biology 19:1651-1660). Suppressing EBVactivities may therefore restore the apoptosis process, which couldexplain the cell death observed in our experiment. Annexin V stainingrevealed a distinct subpopulation of cells with intact cell membrane butexposed phosphatidylserine, suggesting cell death through apoptosis(FIG. 3E-G). Bright field microscopy showed obvious apoptotic cellmorphology (FIG. 3H-I) and fluorescent staining demonstrated drastic DNAfragmentation (FIG. 3J-M). Altogether this evidence suggests restorationof the normal host cell apoptosis pathway after EBV genome destruction.

Complete Clearance Of EBV In A Subpopulation. To study the potentialconnection between cell proliferation arrest and EBV genome editing, wequantified the EBV load in different samples with digital PCR targetingEBNA1. Another Taqman assay targeting a conserved human somatic locusserved as the internal control for human DNA normalization. On average,each untreated Raji cell has 42 copies of EBV genome (FIG. 4A). Cellstreated with a Cas9 plasmid that lacked oriP or sgEBV did not have anobvious difference in EBV load difference from the untreated control.Cells treated with a Cas9-plasmid with oriP but no sgEBV had an EBV loadthat was reduced by ˜50%. In conjunction with the prior observation thatcells from this experiment did not show any difference in proliferationrate, we interpret this as likely due to competition for EBNA1 bindingduring plasmid replication. The addition of the guide RNA cocktailsgEBV1-7 to the transfection dramatically reduced the EBV load. Both thelive and dead cells have >60% EBV decrease comparing to the untreatedcontrol.

Although we provided seven guide RNAs at the same molar ratio, theplasmid transfection and replication process is likely quite stochastic.Some cells will inevitably receive different subsets or mixtures of theguide RNA cocktail, which might affect the treatment efficiency. Tocontrol for such effects, we measured EBV load at the single cell levelby employing single-cell whole-genome amplification with an automatedmicrofluidic system. We loaded freshly cultured Raji cells onto themicrofluidic chip and captured 81 single cells (FIG. 4B). For thesgEBV1-7 treated cells, we flow sorted the live cells eight days aftertransfection and captured 91 single cells (FIG. 4C). Followingmanufacturer's instruction, we obtained ˜150 ng amplified DNA from eachsingle cell reaction chamber. For quality control purposes we performed4-loci human somatic DNA quantitative PCR on each single cellamplification product (Wang J, Fan H C, Behr B, Quake S R (2012)Genome-wide single-cell analysis of recombination activity and de novomutation rates in human sperm. Cell 150:402-412) and required positiveamplification from at least one locus. 69 untreated single-cell productspassed the quality control and displayed a log-normal distribution ofEBV load (FIG. 4D) with almost every cell displaying significant amountsof EBV genomic DNA. We calibrated the quantitative PCR assay with asubclone of Namalwa Burkitt's lymphoma cells, which contain a singleintegrated EBV genome. The single-copy EBV measurements gave a Ct of29.8, which enabled us to determine that the mean Ct of the 69 Rajisingle cell samples corresponded to 42 EBV copies per cells, inconcordance with the bulk digital PCR measurement. For the sgEBV1-7treated sample, 71 single-cell products passed the quality control andthe EBV load distribution was dramatically wider (FIG. 4E). While 22cells had the same EBV load as the untreated cells, 19 cells had nodetectable EBV and the remaining 30 cells displayed dramatic EBV loaddecrease from the untreated sample.

Essential Targets For EBV Treatment. The seven guide RNAs in our CRISPRcocktail target three different categories of sequences which areimportant for EBV genome structure, host cell transformation, andinfection latency, respectively. To understand the most essentialtargets for effective EBV treatment, we transfected Raji cells withsubsets of guide RNAs. Although sgEBV4/5 reduced the EBV genome by 85%,they could not suppress cell proliferation as effectively as the fullcocktail (FIG. 3A). Guide RNAs targeting the structural sequences(sgEBV1/2/6) could stop cell proliferation completely, despite noteliminating the full EBV load (26% decrease). Given the high efficiencyof genome editing and the proliferation arrest (FIG. 2), we suspect thatthe residual EBV genome signature in sgEBV1/2/6 was not due to intactgenomes but to free-floating DNA that has been digested out of the EBVgenome, i.e. as a false positive. We conclude that systematicdestruction of EBV genome structure appears to be more effective thantargeting specific key proteins for EBV treatment.

Additional information such as primer design is shown in Wang and Quake,2014, RNA-guided endonuclease provides a therapeutic strategy to curelatent herpesviridae infection, PNAS 111(36):13157-13162 and in theSupporting Information to that article published online at the PNASwebsite, and the contents of both of those documents are incorporated byreference for all purposes.

Example 2 Targeting Hepatitis B Virus (HBV)

Methods and materials of the present invention may be used to applytargeted endonuclease to specific genetic material such as a latentviral genome like the hepatitis B virus (HBV). The invention furtherprovides for the efficient and safe delivery of nucleic acid (such as aDNA plasmid) into target cells (e.g., hepatocytes). In one embodiment,methods of the invention use hydrodynamic gene delivery to target HBV.

FIG. 10 diagrams the HBV genome. It may be preferable to receiveannotations for the HBV genome (i.e., that identify important featuresof the genome) and choose a candidate for targeting by enzymaticdegredation that lies within one of those features, such as a viralreplication origin, a terminal repeat, a replication factor bindingsite, a promoter, a coding sequence, and a repetitive region.

HBV, which is the prototype member of the family Hepadnaviridae, is a 42nm partially double stranded DNA virus, composed of a 27 nm nucleocapsidcore (HBcAg), surrounded by an outer lipoprotein coat (also calledenvelope) containing the surface antigen (HBsAg). The virus includes anenveloped virion containing 3 to 3.3 kb of relaxed circular, partiallyduplex DNA and virion-associated DNA-dependent polymerases that canrepair the gap in the virion DNA template and has reverse transcriptaseactivities. HBV is a circular, partially double-stranded DNA virus ofapproximately 3200 bp with four overlapping ORFs encoding the polymerase(P), core (C), surface (S) and X proteins. In infection, viralnucleocapsids enter the cell and reach the nucleus, where the viralgenome is delivered. In the nucleus, second-strand DNA synthesis iscompleted and the gaps in both strands are repaired to yield acovalently closed circular DNA molecule that serves as a template fortranscription of four viral RNAs that are 3.5, 2.4, 2.1, and 0.7 kblong. These transcripts are polyadenylated and transported to thecytoplasm, where they are translated into the viral nucleocapsid andprecore antigen (C, pre-C), polymerase (P), envelope L (large), M(medium), S (small)), and transcriptional transactivating proteins (X).The envelope proteins insert themselves as integral membrane proteinsinto the lipid membrane of the endoplasmic reticulum (ER). The 3.5 kbspecies, spanning the entire genome and termed pregenomic RNA (pgRNA),is packaged together with HBV polymerase and a protein kinase into coreparticles where it serves as a template for reverse transcription ofnegative-strand DNA. The RNA to DNA conversion takes place inside theparticles.

Numbering of basepairs on the HBV genome is based on the cleavage sitefor the restriction enzyme EcoR1 or at homologous sites, if the EcoR1site is absent. However, other methods of numbering are also used, basedon the start codon of the core protein or on the first base of the RNApregenome. Every base pair in the HBV genome is involved in encoding atleast one of the HBV protein. However, the genome also contains geneticelements which regulate levels of transcription, determine the site ofpolyadenylation, and even mark a specific transcript for encapsidationinto the nucleocapsid. The four ORFs lead to the transcription andtranslation of seven different HBV proteins through use of varyingin-frame start codons. For example, the small hepatitis B surfaceprotein is generated when a ribosome begins translation at the ATG atposition 155 of the adw genome. The middle hepatitis B surface proteinis generated when a ribosome begins at an upstream ATG at position 3211,resulting in the addition of 55 amino acids onto the 5′ end of theprotein.

ORF P occupies the majority of the genome and encodes for the hepatitisB polymerase protein. ORF S encodes the three surface proteins. ORF Cencodes both the hepatitis e and core protein. ORF X encodes thehepatitis B X protein. The HBV genome contains many important promoterand signal regions necessary for viral replication to occur. The fourORFs transcription are controlled by four promoter elements (preS1,preS2, core and X), and two enhancer elements (Enh I and Enh II). AllHBV transcripts share a common adenylation signal located in the regionspanning 1916-1921 in the genome. Resulting transcripts range from 3.5nucleotides to 0.9 nucleotides in length. Due to the location of thecore/pregenomic promoter, the polyadenylation site is differentiallyutilized. The polyadenylation site is a hexanucleotide sequence (TATAAA)as opposed to the canonical eukaryotic polyadenylation signal sequence(AATAAA). The TATAAA is known to work inefficiently (9), suitable fordifferential use by HBV.

There are four known genes encoded by the genome, called C, X, P, and S.The core protein is coded for by gene C (HBcAg), and its start codon ispreceded by an upstream in-frame AUG start codon from which the pre-coreprotein is produced. HBeAg is produced by proteolytic processing of thepre-core protein. The DNA polymerase is encoded by gene P. Gene S is thegene that codes for the surface antigen (HBsAg). The HBsAg gene is onelong open reading frame but contains three in-frame start (ATG) codonsthat divide the gene into three sections, pre-S1, pre-S2, and S. Becauseof the multiple start codons, polypeptides of three different sizescalled large, middle, and small (pre-S1+pre-S2+S, pre-S2+S, or S) areproduced. The function of the protein coded for by gene X is not fullyunderstood but it is associated with the development of liver cancer. Itstimulates genes that promote cell growth and inactivates growthregulating molecules.

With reference to FIG. 10, HBV starts its infection cycle by binding tothe host cells with PreS1. Guide RNA against PreS1 locates at the 5′ endof the coding sequence. Endonuclease digestion will introduceinsertion/deletion, which leads to frame shift of PreS1 translation. HBVreplicates its genome through the form of long RNA, with identicalrepeats DR1 and DR2 at both ends, and RNA encapsidation signal epsilonat the 5′ end. The reverse transcriptase domain (RT) of the polymerasegene converts the RNA into DNA. Hbx protein is a key regulator of viralreplication, as well as host cell functions. Digestion guided by RNAagainst RT will introduce insertion/deletion, which leads to frame shiftof RT translation. Guide RNAs sgHbx and sgCore can not only lead toframe shift in the coding of Hbx and HBV core protein, but also deletionthe whole region containing DR2-DR1-Epsilon. The four sgRNA incombination can also lead to systemic destruction of HBV genome intosmall pieces.

HBV replicates its genome by reverse transcription of an RNAintermediate. The RNA templates is first converted into single-strandedDNA species (minus-strand DNA), which is subsequently used as templatesfor plus-strand DNA synthesis. DNA synthesis in HBV use RNA primers forplus-strand DNA synthesis, which predominantly initiate at internallocations on the single-stranded DNA. The primer is generated via anRNase H cleavage that is a sequence independent measurement from the 5′end of the RNA template. This 18 nt RNA primer is annealed to the 3′ endof the minus-strand DNA with the 3′ end of the primer located within the12 nt direct repeat, DR1. The majority of plus-strand DNA synthesisinitiates from the 12 nt direct repeat, DR2, located near the other endof the minus-strand DNA as a result of primer translocation. The site ofplus-strand priming has consequences. In situ priming results in aduplex linear (DL) DNA genome, whereas priming from DR2 can lead to thesynthesis of a relaxed circular (RC) DNA genome following completion ofa second template switch termed circularization. It remains unclear whyhepadnaviruses have this added complexity for priming plus-strand DNAsynthesis, but the mechanism of primer translocation is a potentialtherapeutic target. As viral replication is necessary for maintenance ofthe hepadnavirus (including the human pathogen, hepatitis B virus)chronic carrier state, understanding replication and uncoveringtherapeutic targets is critical for limiting disease in carriers.

In some embodiments, systems and methods of the invention target the HBVgenome by finding a nucleotide string within a feature such as PreS1.Guide RNA against PreS1 locates at the 5′ end of the coding sequence.Thus it is a good candidate for targeting because it represents one ofthe 5′-most targets in the coding sequence. Endonuclease digestion willintroduce insertion/deletion, which leads to frame shift of PreS1translation. HBV replicates its genome through the form of long RNA,with identical repeats DR1 and DR2 at both ends, and RNA encapsidationsignal epsilon at the 5′ end. The reverse transcriptase domain (RT) ofthe polymerase gene converts the RNA into DNA. Hbx protein is a keyregulator of viral replication, as well as host cell functions.Digestion guided by RNA against RT will introduce insertion/deletion,which leads to frame shift of RT translation. Guide RNAs sgHbx andsgCore can not only lead to frame shift in the coding of Hbx and HBVcore protein, but also deletion the whole region containingDR2-DR1-Epsilon. The four sgRNA in combination can also lead to systemicdestruction of HBV genome into small pieces. In some embodiments, methodof the invention include creating one or several guide RNAs against keyfeatures within a genome such as the HBV genome shown in FIG. 10.

FIG. 10 shows key parts in the HBV genome targeted by CRISPR guide RNAs.To achieve the CRISPR activity in cells, expression plasmids coding cas9and guide RNAs are delivered to cells of interest (e.g., cells carryingHBV DNA). To demonstrate in an in vitro assay, anti-HBV effect may beevaluated by monitoring cell proliferation, growth, and morphology aswell as analyzing DNA integrity and HBV DNA load in the cells. Thedescribed method may be validated using an in vitro assay. Todemonstrate, an in vitro assay is performed with cas9 protein and DNAamplicons flanking the target regions. Here, the target is amplified andthe amplicons are incubated with cas9 and a gRNA having the selectednucleotide sequence for targeting. As shown in FIG. 11, DNAelectrophoresis shows strong digestion at the target sites.

FIG. 11 shows a gel resulting from an in vitro CRISPR assay against HBV.Lanes 1, 3, and 6: PCR amplicons of HBV genome flanking RT, Hbx-Core,and PreS1. Lane 2, 4, 5, and 7: PCR amplicons treated with sgHBV-RT,sgHBV-Hbx, sgHBV-Core, sgHBV-PreS1. The presence of multiple fragmentsespecially visible in lanes 5 and 7 show that sgHBV-Core and sgHBV-PreS1provide especially attractive targets in the context of HBV and that useof systems and methods of the invention may be shown to be effective byan in vitro validation assay.

Example 3 Varicella-Zoster Therapy

Shingles—sometimes known as herpes zoster, zoster, chickenpox virus,human herpesvirus type 3 (HHV-3), or zona—is a viral diseasecharacterized by blisters and rash that come with great pain to thepatient, which pain lasts as much as four to six weeks. The rash oftenappears in a characteristic stripe on a side of the body. Some peopledevelop ongoing nerve pain which may last for months or years, acondition called postherpetic neuralgia.

Shingles and postherpetic neuraligia are associated with a reactivationof varicella zoster virus (VZV) within a person. VZV only effects humansand is the cause of chickenpox in young people. VZV infects the nerves,and causes a wide variety of symptoms. After the primary infection(chickenpox), the virus goes dormant in the nerves, including thecranial nerve ganglia, dorsal root ganglia, and autonomic ganglia. Manyyears after the patient has recovered from chickenpox, VZV canreactivate to cause a number of neurologic conditions.

Compositions that include a vector comprising a gene for a nuclease, asequence that targets the nuclease to a genome of a virus, and apromoter that promotes transcription from the vector within cells of aspecific type may be used to treat or prevent conditions such asshingles or postherpetic neuraligia. Using methods and compositions ofthe invention to treat an infection such as by varicella zoster virusmay include delivering the nuclease to specific cell types such asneurons.

Methods of the invention include targeting vectors to neurons and othercell types. Any suitable targeting method can be used. For example,targeting can include microsurgery or using the cytomegalovirus (CMV)promoter (11) or the Rous sarcoma virus (RSV) enhancerypromoter (pRcRSV,Invitrogen)as described in Glatzel et al., 2000, Adenoviral andadeno-associated viral transfer of genes to the peripheral nervoussystem PNAS 97(1):442-447, incorporated by reference. For the promoters,see also Liu et al., 2004, CMV enhancer/human PDGF-beta promoter forneuron specific transgene expression, Gene Ther 11(1):52-60,incorporated by reference. For additional discussion of targetingneurons, see Sapunar et al., 2012, Dorsal root ganglion—a potential newtherapeutic target for neuropathic pain, J Pain Res 5:31-38,incorporated by reference. Delivering an active targetable nucleasespecifically to neurons (and preferably the DRG) may be used to treat orprevent shingles or postherpetic neuralgia. In the nerve cells, thepromoter causes the expression of the genes selectively within the nervecells. The promoter may be, for example, a cytomegalovirus promoter, aRous sarcoma virus promoter, or a platelet-derived growth factor (PGDF)promoter.

The targeting sequence may be designed to target a regulatory element inthe genome of the virus and preferably lacks any exact match in a humangenome. The targeting sequence may be a portion of the vector that codesfor a gRNA. The nuclease may be a cas9 endonuclease. In someembodiments, the sequence is within a clustered regularly interspacedshort palindromic repeats (CRISPR) region within the vector, and theCRISPR region encodes a plurality of guide RNAs that match a pluralityof targets within the genome of the virus.

1-18. (canceled)
 19. A composition for treating a viral infection, thecomposition comprising: a vector that includes a gene for a Cas9endonuclease, a sequence that encodes one or more guide RNAs that targetthe nuclease to nucleic acid from a genome of the Varicella-zostervirus, and a regulatory element that causes the gene for the Cas9endonuclease to be active within a cell that is infected by the virus.20. The composition of claim 19, wherein the regulatory element is froma genome of the virus.
 21. The composition of claim 20, wherein theregulatory element is one selected from the group consisting of apromoter and an origin of replication. 22-29. (canceled)
 30. Thecomposition of claim 19, wherein the one or more guide RNAs are designedto target a regulatory element in the genome of the virus. 31.(canceled)
 32. A composition for treating a viral infection, thecomposition comprising: a vector comprising a gene for a Cas9endonuclease, a sequence that targets the nuclease to a genome of theVaricella-zoster virus, and a promoter that promotes transcription fromthe vector within cells of a specific type.
 33. The composition of claim32, wherein the cells are nerve cells and the promoter further causesthe expression of the gene selectively within the nerve cells.
 34. Thecomposition of claim 33, wherein the promoter comprises acytomegalovirus promoter, a Rous sarcoma virus promoter, or aplatelet-derived growth factor (PGDF) promoter.
 35. (canceled)
 36. Thecomposition of claim 33, wherein the sequence is designed to target aregulatory element in the genome of the virus and lacks any exact matchin a human genome.
 37. (canceled)
 38. The composition of claim 36,wherein the sequence is within a clustered regularly interspaced shortpalindromic repeats (CRISPR) region within the vector, wherein theCRISPR region encodes a plurality of guide RNAs that match a pluralityof targets within the genome of the virus.
 39. The composition of claim32, wherein the promoter promotes transcription within the peripheralnervous system.
 40. The composition of claim 39, wherein the vectorcomprises an adenoviral vector or a rAAV-based vector.